Influenza A viruses are divided into subtypes on the basis of the antigenicity of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA); influenza viruses bearing 15 HA and 9 NA subtypes have been isolated from birds, but only H1N1, H2N2, and H3N2 subtype viruses have circulated widely and caused epidemic disease in humans in the last century. Aquatic birds serve as a reservoir from which new subtypes of influenza A viruses enter the human population. In the last 10 years, human infections with avian influenza viruses (AIV) of three subtypes, H7, H5 and H9, have been detected on several occasions, accompanied in each case by contemporaneous outbreaks of disease in poultry. On several occasions since 1997, there have been serious outbreaks caused by a highly pathogenic avian influenza H5N1 viruses in Asia. The most recent H5N1 outbreak in poultry began in late 2003 and affected at least 10 countries in Asia. Recent reports indicate that H5N1 viruses have been isolated from migratory and wild birds in Asia, Europe and Africa. Human cases of H5N1 infection have been reported since December 2003 in 5 countries with a total of 232 cases and 134 deaths as of August 1, 2006. There are several potential strategies for the development of vaccines to protect humans against influenza viruses, including formalin inactivated whole or split virus, HA subunit, and live attenuated vaccines. Live attenuated vaccines generally induce broadly cross-reactive protection, which may be a useful feature in the event of a pandemic if a vaccine generated from the actual pandemic strain is not available. The goal of our program is to generate candidate live attenuated reassortant influenza virus vaccines against a range of influenza A subtypes that have pandemic potential and to evaluate these vaccines in preclinical studies and clinical trials. The vaccine viruses will contain the hemagglutinin (HA) and neuraminidase (NA) genes of a selected avian influenza virus with pandemic potential and the attenuating genes from the A/Ann Arbor/6/60 cold adapted (A/AA/6/60 ca) donor virus. The cold-adapted (ca) influenza virus A/Ann Arbor/6/60 (AA) (H2N2) has been developed as a live attenuated vaccine seed virus that exhibits cold-adaptation, temperature-sensitive (ts), and attenuation (att) phenotypes which are specified by mutations in the genes encoding the non-surface glycoprotein genes, i.e., the genes encoding the internal protein genes. Reassortant H1N1 and H3N2 human influenza A viruses with the six internal gene segments of the AA ca virus have been repeatedly demonstrated to bear the ts and att phenotypes and extensive evaluation in humans has proven them to be attenuated and safe as live virus vaccines. This approach has been licensed for general use for interpandemic influenza A and B virus infections. Live attenuated vaccines must be able to replicate to levels that elicit a protective immune response without causing disease in the host so a balance between attenuation, infectivity and immunogenicity must be achieved. An optimal public health response in the event of a potential pandemic requires that vaccines be available to prevent infection with minimum delay and an important approach to pandemic preparedness is to generate and evaluate candidate vaccines against influenza A subtypes that are recognized to have pandemic potential, prior to their actual emergence of a pandemic virus. We generated a candidate H9N2 influenza vaccine by genetic reassortment; the vaccine strain contains the hemagglutinin and neuraminidase genes from an avian H9N2 influenza virus and six internal gene segments from the AA ca virus. Based on promising preclinical data in mice and ferrets, a clinical lot of this vaccine was generated and a Phase I clinical trial of the safety and immunogenicity of the vaccine for healthy adults was undertaken under an IND. Analysis of laboratory results from the clinical trial is under way. In order to generate candidate vaccines against H5N1 viruses that have caused human infections in Asia in 1997, 2003 and 2004, LID scientists collaborated with scientists from Medimmune, Inc., under a CRADA and applied plasmid based reverse genetics, a technique in which infectious virus can be recovered from cells co-transfected with plasmids expressing each of the 8 influenza gene segments, to generate reassortant viruses that contain the hemagglutinin and neuraminidase genes from H5N1 influenza viruses and six internal gene segments from the AA ca virus. We removed the virulence motif of multiple basic amino acid motifs in the hemagglutinin gene of the highly pathogenic H5N1 influenza virus that are associated with pathogenicity in poultry. The candidate H5N1 vaccine viruses possessed the ts and att phenotypes specified by the internal protein genes of the AA ca virus. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of vaccine, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wild-type H5N1 viruses isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. Based on promising preclinical data in mice and ferrets, a clinical lot of the 2004 H5N1 ca vaccine was generated and a Phase I clinical trial of the safety and immunogenicity of the vaccine for healthy adults was undertaken under an IND. The clinical trial is in progress.